Cell--cell interactions play an important role in regulating cell fate decisions and pattern formation during the development of multicellular organisms. One of the evolutionarily conserved pathways that plays a central role in local cell interactions is mediated by the transmembrane receptors encoded by the Notch (N) gene of Drosophila, the lin-12 and glp-1 genes of C. elegans, and their vertebrate homologs (reviewed in Artavanis-Tsakonas, S., et al. (1995) Notch Signaling. Science 268, 225-232). collectively hereinafter referred to as NOTCH receptors. Several lines of evidence suggest that the proteolytic processing of NOTCH receptors is important for their function. For example, in addition to the full length proteins, antibodies against the intracellular domains of NOTCH receptors have detected C- terminal fragments of 100-120 kd (hereafter referred to as 100 kd fragments); see e.g. Fehon, R. G., et al. (1990). Cell 61, 523-534; Crittenden, S. L., et al. (1994). Development 120, 2901-2911; Aster, J., et al. (1994) Cold Spring Harbor Symp. Quant. Biol. 59, 125-136; Zagouras, P., et al.(1995). Proc. Natl. Acad. Sci. USA 92, 6414-6418; and Kopan, R., et al. (1996). Proc. Natl. Acad. Sci. USA 93, 1683-1688. However, the mechanism(s) of NOTCH activation have been hitherto largely unknown.
During neurogenesis, a single neural precursor is singled out from a group of equivalent cells through a lateral inhibition process in which the emerging neural precursor cell prevents its neighbors from taking on the same fate (reviewed in Simpson, P. (1990).
Development 109, 509-519). Genetic studies in Drosophila have implicated a group of "neurogenic genes" including N in lateral inhibition. Loss-of-function mutations in any of the neurogenic genes result in hypertrophy of neural cells at the expense of epidermis (reviewed in Campos-Ortega, J. A. (1993) In: The Development of Drosophila melanogaster M. Bate and A. Martinez-Arias, eds. pp. 1091-1129. Cold Spring Harbor Press.). We disclose herein a new neurogenic gene family, kuzbanian (kuz) (Rooke, J., Pan, D. J., Xu, T. and Rubin, G. M. (1996). Science 273, 1227-1231). Members of the disclosed KUZ family of proteins are shown to belong to the recently defined ADAM family of transmembrane proteins, members of which contain both a disintegrin and metalloprotease domain (reviewed in Wolfsberg, T. G., et al. (1995). J. Cell Biol. 131, 275-278, see also Blobel, C. P., et al. (1992). Nature 356, 248-252, 1992; Yagami-Hiromasa, T., et al. (1995). Nature 377, 652-656; Black, R. A., et al. (1997). Nature 385, 729-733, 1997; and Moss, M. L., et al. (1997). Nature 385, 733-736).
We further disclose herein various engineered mutant forms of native KUZ proteins and their activities. We show that mutant KUZ proteins lacking protease activity interfere with endogenous KUZ activity and function as dominant negatives (indicating that the protease activity of native KUZ is essential to its biological functions) and that dominant negatives can perturb lateral inhibition during neurogenesis and result in the overproduction of primary neurons. We also show that proteolytic processing of NOTCH in embryos to generate the 100 kd species fails to occur in the kuz mutant embryo and expression of dominant negatives in imaginal discs or tissue culture cells blocks NOTCH processing (indicating that the primary NOTCH translation product is proteolytically cleaved by native KUZ proteins as part of the normal biosynthesis of a functional NOTCH receptor).